Project Summary/Abstract (30 lines of text) Despite our knowledge about DNA damage responses, we know significantly less about the cellular pathways activated selectively with mitochondrial DNA (mtDNA) damage. In fact, when cells are exposed to DNA damaging drugs, the responses observed are likely a consequence of damage to both the nuclear DNA and mtDNA. However, separating the outcome of nuclear DNA damage versus mtDNA damage is difficult because of the challenges of exposing only the mitochondria and not the nucleus to DNA damage in cells. To selectively expose the mitochondria but not the nucleus to DNA damaging drugs, we utilized primary neurons as their cell body (containing the nucleus) and axons (containing mitochondria but not the nucleus) are spatially distinct. We use microfluidic chambers to culture the neurons, which allowed us to expose only the axons and not the cell bodies to DNA damaging drugs such as cisplatin. Addition of cisplatin exclusively to the axons in microfluidic chambers induced mtDNA damage in axonal mitochondrial without any DNA damage to the nucleus. Importantly, cisplatin addition induced widespread axon degeneration. No degeneration was seen in the cell body or neuronal regions that were not exposed to cisplatin. Similar selective degeneration of axons was also observed when axons were exposed to other DNA damaging drugs such as the nucleoside analog d4T or the topoisomerase I inhibitor camptothecin. In thisR21 proposal, we will focus on defining the molecular mechanism of axon degeneration induced with mtDNA damage. While nuclear DNA damage is recognized to activate a p53-depedent apoptotic cell death pathway, virtually nothing is known about the specific cell degeneration pathway activated with mtDNA damage. Thus, in Aim 1 we will investigate whether key proteins of the apoptotic pathway are required for axon degeneration in response to mtDNA damage. Specifically, we will evaluate mtDNA damage-induced axon degeneration in wildtype neurons or neurons deficient in p53, Bax, Apaf-1 Caspase-9 or Caspase-3. In addition to apoptosis, axon degeneration has been studied in the context of pruning and axotomy-induced Wallerian degeneration. Thus, in Aim 2, we will examine whether axon degeneration after mtDNA damage is mediated by either the pruning (using Caspase-6-deficient neurons) or Wallerian (using Sarm-1-deficient neurons) axon degeneration pathways. Our focus in Aim 3 is on mitophagy as damaged mitochondria are recognized to be targeted for degradation via Parkin-mediated degradation. While mitophagy is generally beneficial to neurons, excess mitophagy could also be detrimental, particularly in situations of acute mitochondrial damage. We will utilize Parkin-deficient neurons to critically evaluate whether inhibition of mitophagy accelerates or reduces the kinetics of axon degeneration with acute mtDNA damage. Our approach of inducing mtDNA damage in axons using the neuronal microfluidic chamber model has broad relevance, as it will help define the nuclear DNA damage-independent mechanisms of degeneration.